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530
Human Nonmuscle Myosin Heavy Chains Are
Encoded by Two Genes Located on
Different Chromosomes
Michael Simons, Mary Wang, 0. Wesley McBride, Sachiyo Kawamoto,
Katsutoshi Yamakawa, David Gdula, Robert S. Adelstein, and Lawrence Weir
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We report the cloning of cDNAs encoding two different human nonmuscle myosin heavy chains
designated NMMHC-A and NMMHC-B. The mRNAs encoding NMMHC-A and NMMHC-B
are both 7.5 kb in size but are shown to be the products of different genes, which are localized
to chromosome 22q11.2 and chromosome 17p13, respectively. In agreement with previously
reported results using avian tissues, we show that the mRNAs encoding the two myosin heavy
chain isoforms are differentially expressed in rat nonmuscle and muscle tissues as well as in a
number of human cell lines. The cDNA sequence encoding the 5' portion of the NMMHC-A
isoform completes the previously published 3' cDNA sequence encoding a human myosin heavy
chain, thus providing the cDNA sequence encoding the entire NMMHC-A amino acid sequence.
Comparison of this sequence to cDNA clones encoding the amino-terminal one third of the
NMMHC-B sequence (amino acids 58-718) shows them to be 89% identical at the amino acid
level and 74% identical at the nucleotide level. (Circulation Research 1991;69:530-539)
M yosin is a ubiquitous cytoskeletal protein
present in all eukaryotic cells. Although
best studied in tissues that manifest specialized contractile activity, such as cardiac and skeletal muscle, myosin in nonmuscle cells has been
implicated in processes as diverse as cytokinesis, cell
motility (for reviews, see References 1-3), secretion,4
and capping.5 There is now compelling genetic evidence that supports a role for nonmuscle myosin in
cytokinesis, because cells that lack most of a particular myosin isoform lose their ability to divide.6,7
In vertebrates, the conventional myosin isoforms
consist of a pair of heavy chains (200 kDa) and two
pairs of light chains (15-28 kDa). In addition to the
480 kDa myosin isoforms, vertebrate intestinal brush
border epithelial cells, similar to Acanthamoeba and
Dictyostelium, contain a smaller myosin isoform (110
kDa) that preserves many of the functional properties of the globular amino terminal region of the
From the Laboratory of Biochemistry (M.W., O.W.M.), National
Cancer Institute, and the Laboratory of Molecular Cardiology (M.S.,
S.K, KY., D.G., R.S.A.), National Heart, Lung, and Blood Institute,
National Institutes of Health, Bethesda, Md.; and St. Elizabeth's
Hospital (L.W.), Department of Cardiology, Boston, Mass.
The sequences reported in this paper have been deposited in the
GenBank data base (accession Nos. M69180 and M69181).
Address for correspondence: Robert S. Adelstein, MD, Laboratory of Molecular Cardiology, National Institutes of Health,
Building 10, Room 8N-202, Bethesda, MD 20892.
Received January 8, 1991; accepted April 15, 1991.
myosin heavy chain (MHC) but lacks the carboxyterminal rod region (for review, see Reference 8).
Skeletal and cardiac MHCs exist as multigene families with a large number of isoforms encoded by different genes. The two different cardiac MIC isoforms
manifest different rates of ATP hydrolysis9 and may
serve different physiological functions. Less is known
about the MHC isoforms present in vertebrate smooth
muscle and nonmuscle cells. At present, two different
smooth muscle MHC isoforms have been identified at
the protein level,10 and the isolation of cDNA1112 and
genomic clones'3 suggests that at least three MHC
isoforms can be generated by alternative splicing of
mRNA. For vertebrate nonmuscle MHCs (NMMHCs),
the existence of isoforms first was suggested on the
basis of peptide maps by Burridge and Bray.'4 Recently, the sequence for two cDNA clones (2.8 and 0.9
kb) encoding the same region of the NMMHC in
chicken fibroblasts, but that showed differences
throughout this sequence, was reported.15 These authors suggested that these two clones are encoded by
different genes. The 0.9 kb clone is present in the
nucleotide sequence of the cDNA encoding the entire
NMMHC from chicken intestinal epithelial cells reported previously,16 which we refer to as NMMHC-A.
The first mammalian NMMHC cDNA clones were
obtained from a human macrophage and fibroblast
library by Saez et al,'7 and comparison of these sequences with the avian sequence identified the human
clones as the NMMHC-A isoform.
Simons et al Nonmuscle Myosin Heavy Chains
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One of the most interesting aspects of nonmuscle
myosin function is the role it may play in smooth
muscle cell proliferation. Proliferation of vascular
smooth muscle is a cardinal feature of atheroscleroSiS.18 In addition, it has been implicated in the
development of restenosis after angioplasty of vascular lesions,19 in the failure of venous bypass grafts,20
and in the vascular response to hypertension.21 Proliferating vascular smooth muscle undergoes a striking phenotypic change: instead of spindle-shaped
cells capable of contractile activity, but incapable of
division, one encounters polygonal actively secreting
cells incapable of contraction, but capable of cytokinesis. On the biochemical level, a number of alterations occur as well. One of the most striking is an
almost complete replacement of smooth muscle
MHC with a nonmuscle isoform.22 This change in
MHC phenotype has been observed in proliferating
smooth muscle in culture22,23 as well as in vivo.24
Recently, we have demonstrated the expression of
nonmuscle myosin in restenotic lesions in human
coronary as well as peripheral arteries.25 The appearance of the nonmuscle isoform correlates with the
newly acquired ability of smooth muscle cells to
divide and disappears when cells return to their
contractile phenotype. These observations suggest
that nonmuscle myosins may play a pivotal role in
smooth muscle proliferation and that understanding
their role may shed light on the pathogenesis of a
number of important vascular diseases.
In this paper we present information on the cloning
and sequence of two human NMMHC cDNAs. The
sequence of one group of cDNA clones encodes the 5'
portion of NMMHC-A. The second group of clones
encodes a different NMMHC isoform, which we refer
to as NMMHC-B. We show that these two isoforms
are encoded by two different genes. Chromosomal
mapping using hybrid panels and in situ hybridization
showed that these genes are located on different
chromosomes. The gene encoding NMMHC-A was
localized by in situ hybridization to chromosome
22q11.2. The gene encoding NMMHC-B was localized
to chromosome 17p13. This chromosome band contains at least one and possibly four genes encoding the
skeletal muscle MHCs.26-30
Materials and Methods
cDNA Cloning
cDNA clones were isolated from a lambda gtlO
library (Clontech Laboratories, Inc., Palo Alto, Calif.) and a lambda ZAP II library (Stratagene Inc., La
Jolla, Calif.). Both libraries were constructed from
human T lymphocyte (Jurkat) mRNA. The lambda
gtlO library was screened initially with a 900 bp
cDNA probe encoding the 5' region of a chicken
NMMHC-A.16 Subsequently, a second NMMHC-A
probe from the 5' end, as well as human probes
isolated during the cloning process, were used to
screen the library. The lambda ZAP II library was
screened as outlined in "Results." Probes were la-
531
beled by random priming to a specific activity of 108
cpm/,ug using a kit (Prime Time C, United States
Biochemical Corp., Cleveland, Ohio). Hybridization
was carried out at 42°C in 40% formamide and 10%
dextran sulfate. The stringency of the final wash
using chicken cDNA probes was 0.2x SSC at 50°C
and using human cDNA probes was 0.1 x SSC at
65°C. Phage inserts were subcloned either into
pTZ19R (Pharmacia LKB Biotechnology, Piscataway, N.J.) or pGEM-3Zf(-) (Promega Corp., Madison, Wis.) vectors. In the case of clones obtained
from the lambda ZAP II library, in vivo excision of
clones was carried out as outlined by Stratagene.
Sequence Analysis
Double-stranded DNA was sequenced using a
Sequenase kit (United States Biochemical Corp.). In
some cases, restriction enzymes were used to make
deletions in the original subclones that then were
used as sequencing templates. Synthetic oligonucleotide primers were used to extend the nucleotide
sequence. Sequence data was analyzed with the use
of MICROGENIE software (Beckman Instruments,
Inc., Fullerton, Calif.).
mRNA Analysis
Human RNA was prepared from cultured T
lymphocytes by the method of McDonald et a131 or
Chomczynski and Sacchi.32 Total rat RNA from a
number of tissues was prepared by the latter
method, as was total RNA from various human cell
lines. Human cell lines, with the exception of Jurkat
and A-431, were obtained from the American Type
Culture Collection (ATCC) (Rockville, Md.), and
cells were grown as per the instructions. A-431 cells
were a gift from Dr. Stuart Aaronson (National
Cancer Institute) and were grown in Dulbecco's
modified Eagle's medium and 10% fetal bovine
serum. Jurkat cells were obtained from Dr. Warren
Leonard (National Institute of Child Health and
Development) and were grown in RPMI-1640 medium and 10% fetal bovine serum. RNA samples
were analyzed in a denaturing formaldehyde agarose (1%) gel according to a standard protocol,33
and capillary transfer to nylon membranes was
carried out according to manufacturers' recommendations. Hybridization was carried out in the presence of 10% dextran sulfate and 40% formamide at
42°C for 12-16 hours with a final wash in 0.1 x SSC
at 65°C or 0.2x SSC at 60°C for human samples.
For analysis of rat mRNA, hybridization was carried out as above, but the stringency of the final
wash was 0.5 x SSC or 0.2 x SSC at 60°C.
Preparation of Probes for Chromosome Localization
Cloned cDNA and genomic fragnents were used.
The cDNA probes are described in this paper. A 350
bp genomic probe was obtained after EcoRI digestion
of a 17 kb genomic clone obtained from a human
placenta Charon 4A library (ATCC; Simons, Weir,
Adelstein, unpublished data). This genomic clone was
selected using a human lymphocyte cDNA probe, HL1.16 The 350 bp genomic intronic fragment is 5' to an
532
Circulation Research Vol 69, No 2 August 1991
836 ROD 1961
Ifa 11
AI
HEAD (S1)
a
-
i
MHC-A
400
AA
NT
225
-156
675
L
AA
NT
,__~~------721
1606
589
2163
(+707)
376
172
344
¢
FIGURE 1. cDNA clonesofhumannonchain (NMMHC)
fb302 and bZ801 and
Z802 encode the human NMMHC-A
and clones 0707, 756, 758, 759, and 760
encode the human NMMHC-B. Clone
pNMHCM2 is from Saez et al.17Dashed
lines represent areas that have not
been sequenced. AA, amino acid; NT,
nucleotide.
t&.L
-,.'.302pNMHCM2
... muscle myosin heavy
isofonns. Clones
2146
1676
58
MHC-B
715
H-|(+302)
-- -
711
| ~~(+758)
710
(+759)
395
+,ao
718
(+756, +760)
2154
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
DNA Isolation and Filter Hybridization
DNA was isolated from hybrid cell lines, digested
with EcoRI, and size-fractionated by agarose (0.7%)
gel electrophoresis. After partial depurination, the
fragments were transferred to positively charged
nylon membranes in 0.4 M NaOH. The membranes
were hybridized at high stringency (allowing
10%
sequence divergence) at 42°C with 32P-labeled probes
in 50% formamide containing 5x SSPE, 5x Denhardt's solution, 10% dextran sulfate, 0.2% sodium
dodecyl sulfate, and sheared denatured herring
sperm DNA at 200 ,ug/ml. Membranes were washed
at 55°C in 0.1 x SSC containing 0.2% sodium dodecyl
sulfate. The membranes were used repeatedly after
removal of the probe in 0.4 M NaOH, neutralization,
and prehybridization with carrier DNA.
(0.05 ,ug/ml) present during the final 20 minutes. The
cells were centrifuged, swollen, and fixed, and airdried metaphase spreads were prepared by standard
procedures.38 After treatment with RNase A (100
,ug/ml) for 1 hour at 37°C, the chromosomal DNA
was denatured in 70% formamide and 2x SSC at
70°C or in 0.07N NaOH in 64% ethanol at 25°C for 3
minutes.39,40 Radiolabeled probes (specific activity,
3 x 107 cpm/,ug) were prepared by nick translation of
recombinant plasmid DNAs with [3H]dTTP and
[3H]dCTP. The probe was mixed with hybridization
solution (50% formamide, 5% dextran sulfate, 2x
Denhardt's solution, 2x SSC, 5 mM EDTA, 20 mM
sodium phosphate, pH 6.4, and 200 ,g/ml sheared
herring sperm carrier DNA), heat denatured, applied
to slides (3 x 105 cpm/slide in 25 ,ul), and hybridized for
20 hours at 42°C. Nonspecifically bound probe was
removed by washing in 50% formamide/2x SSC (pH
7.0) for 10 minutes at 42°C and in 2 x SSC at 42°C.
The slides were coated with a 50% solution of NTB2
nuclear track emulsion (Eastman Kodak Co., Rochester, N.Y.) and stored dessicated at 4°C for 9 days
before developing, staining (0.25% Wright stain), and
photographing. The slides were destained and
G-banded with 0.03% trypsin and 0.12% EDTA41 or
replication banded37 by staining with 33258 Hoechst
(150 ,ug/ml) for 30 minutes and exposure to UV
illumination for 30 minutes after rinsing. The slides
again were stained with Wright stain, and the same
metaphase spreads were rephotographed.
In Situ Hybridization
Experiments were performed using peripheral
blood lymphocytes from a normal male (46,XY) that
were cultured for 72 hours at 37°C in RPMI-1640
supplemented with 15% fetal bovine serum, phytohemagglutinin (0.5 ,ug/ml), and antibiotics. Cultures
were synchronized by addition of 100 ,ug/ml
5-bromo-2'-deoxyuridine37 or 10` M methotrexate38
for 17 hours before washing and resuspension in
fresh medium containing 10-5 M thymidine and
incubation for an additional 5.5 hours with colcemid
Results
cDNA Cloning and Sequencing
A 900 bpAva I cDNA probe encoding the first 300
amino acids of the chicken intestinal epithelial cell
NMMHC16 was used to obtain a single clone from a
human T lymphocyte lambda gtlO library (0302,
Figure 1). The clone contains a 1,356 bp insert that
consists of 156 nucleotides of 5' untranslated cDNA
and 1,200 nucleotides encoding the first 400 amino
acids of a NMMHC. The location of the translation
exon encoding the reactive thiols found in the myosin
globular head. All fragments were purified by gel
electrophoresis and labeled with dCTP (a-32P) by random oligonucleotide primed synthesis before use.
Cell Hybrids
The human and rodent parental cells, fusion procedure, and isolation and characterization of humanmouse and human-Chinese hamster somatic cell hybrids have been described.34-36 Briefly, hybrid cells
were analyzed for the presence of all human chromosomes except Y by standard isoenzyme analyses, as well
as by Southern analysis with probes from previously
localized genes, and frequently by cytogenetic analysis.
Simons et al Nonmuscle Myosin Heavy Chains
A
gtcctgcita
ELA -19
533
LysLysGi.Ar*AuuTbrAspGi.AI SmrM.tPreAspAsmThrAlaAIaGInLysVu 1 374
1063 AAGAAGGAGCGGAACACTGACCAGGCGTCCATGCCCGACAACACAGCTGCCCAAAAGGTG
A-A-T--- -T-A-T
A-A--T-T-G-G-C-C
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1HU 1072 -A
AA
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52
HiMtAiiGiuGIiAIoAIaAspLysTTyrLiTyrValAsupLyuAnPhel Ie 17
taigtacicATGGCACAGCAAGCTGCCGATAAGTATCTCTATGTGGATAAAAACTTCATC
AsiAuuPriLoiAlGlinAluAspTrpAliAlLyuLyuLueoVuITrpVaIPriSirAup 37
AACAATCCGCTGGCCCAGGCCGACTGGGCTGCCAAGAAGCTGGTATGGGTGCCTTCCGAC
SerArHisLsuLeGlmlGIyeAsnVaIThrAspPhsTbrArgGIyIIluuTbrPrsArgllu 394
1123 TCCCATCTCTTGGGTATCAATGTGACCGATTTCACCAGAGGAATCCTCACCCCGCGCATC
TC-T-G-G
TG-G-T-TC-G-CC
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CVs - - - - Met - - HMGtG1 - - - Ag - - - . - - - 397
nICA
LyuVaIGIArgAspTyrVa1GInLysAIaGIuThrLysGIuGInAIaAspPheAIaI Ie 414
1193 AAGGTGGGACGGGATTACGTCCAGAAGGCGCAGACTAAAGAGCAGGCTGACTTT6CCATC
C-C-A -C-T---A-A-C
A
A-TAG-A
C
- - - - - - - - - - - - - - - - - - - Val 417
ECB 1192 -
IHGA
112
LysSirGi yPhuGluPrsAliaSerLunLyVGiGliVolGlt6iGArgGlNHiuVilGin 57
AAGAGTGGCTTTGAGCCAGCCAGCCTCAAGGAGGAGGTGGGCGAGAGAGGCCATGTGGAG
MUCA
1243
GIuAI6LaLAIl,LsAlaThrTyrGIuArgH.tPhaArgTrpLemVaIleLArI ImAui 434
GAGGCCTTGGCCAAGGCGACCTATGABCBGATGTTCCGCTGGCTGBTGCTGCGCATCAAC
MfU 1252 -A-A-A-A-T
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HHA
172
LiiVaiGliAtnfllyLyVlLiVulAiALyiAsipAipl InGlnLynHietAiPri 77
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ECI
232
PriLyaPhuSerLytValGlmAspMiltAl iiLiuThrCyiLiiAsnoGlAlnSerVil 97
CtCAAGTTCTCCAAGGTGGAGGACATGGCAGAGCTCACGTGCCTCAACGAAGCCTCGGTG
T
T-C-T
-T-T
AT-G-A-T-G-T
-
-
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-
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T
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292 CTGCACAACCTCAAGGAGCG TTACTACTCAGGGCTCATCTACACCTATTCAGGCCTGTTC
EC
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T-A-CT-A-T-T-G--T-C
T-G
-
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A-A-T
AC-T
352
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TGTGTGGTCATCAATCCTTACAAGAACCTGCCCATCTACTCTGAAGAGATTGTGGAAATG
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
GA-T-A-T
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T-T-A-T
A-T-A-C
.--
-
457
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-
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-
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412
MA
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472
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C-T-T
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532
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TyrArgSurMetMitiGliApArgGliAnpGhiSurl iLeuCynThrGlyGliSerGly 177
AlaGlyLysThrGiAuiThrLyiLysVallii GInTyrLouAiaTyrValAhiSurSir 197
-
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592
ECU
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661
iG 1Gi
CAGGGCGAGCTGGAGCGGCAGCTGCTGCAGGCC
CACAAGAGCAAGAA6GAC
-TT-A-A
CATAATATT-CT-G-A-T-A
-T-AG-A-GA
- - G1VAr, - - NihiAuliPri - - - - - - - - - - 217
MIHC
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-
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Sir
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-
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703 TTCGGCAAATTCATTCGCATCAACTTTGATGTCAATGGCTACATTGTTGGAGCCAACATT
1: 712 -T
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6G
.
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763
MIC 7
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623
ECU 832
MIC;A
M3
ECU 832
-
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Thr
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-
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-
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257
GAGACTTATCTTTTGGAGAAATCTCGTGCTATCCGCCAAGCCAAGGAAGAACGGACCTTC
-A-A-C-C-A-B
G-T-T- A-A-T
T-T-T
- - - - - - - - - - Vol - - - - Asp - - - - m
HNiuI11PhuTyrTyrLeuleuSerrGlyAIaulyBi6iuL nLysThrAspLuuLnLeun 294
CACATCTTCTATTATCTCCTGTCTG666CTGGAGAGCACCTGAAGACCGATCTCCTGTTG
-A-A-A
-T-T-CC-6T-BT-A
A-T-T-T-G-TC-T
- - -i - - - - - - - - - - Ser - - - - 297
BluPruTVrAuLyusTyrAr,PbeleLSurAuGIyNHiuVaiTbri IuPruGIVBiuGio 314
GAGCCGTACAACAAATACCGCTTCCTGTCCAAT6GACACGTCACCATCCCCGGGCAGCAG
CT-TA-TC-T-T-G-A
A
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EU 1792
EA
1B43
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497
-
-
-
-
517
BiiiuHilyLuuLuoArVul IiSirGlrVuiLunGluLiuGiAsul iiViPhu 354
1003 GAGCAAATG6GCCTGCTGCGGGTCATCTCAGG6GTTCTTCAGCTCGGCAACATCGTCTTC
r
-
-
EC
577
-
-
-
-
W597
-
614
617
934
937
AIPhiLyiThrAr,LyoGlyMetPheArgThrVail yGtaLeuTyrLyoGi.GinLin 954
1993 GCCTTCAAGACGCGGAAGGGCATGTTCCGCACTGTGGGGCAGCTTTACAAGGAGCAGCTG
1912 -A-AT-A-CAA
- Tyr - - Lys
-
-
-
-
-
-
A-ATCT-C
A-C
T-T-C-T
-
-
-
-
-
-
-
Ser
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957
AAAliLsLiiHtAiiSorLoArgAsiThrAunPriAioPbiVilArgCyul ei limPri 674
1993 GCCAAGCTGATGGCATCGCTGAGGAACACGAACCCCAACTTTGTCCGCTGCATCATCCCC
T
1972 A
CC-CA-T-CC-A-C
T-A
T-T-T
- Thr 677
AsnHi s61GlViLytAIaGlyLyslouAspPrtNiiLeuVIlLeuAspGinLuiArgCyu 694
Thy - - low
2323 AACCACGAGAAGAAGGCCGGCAAGCTGGACCCGCATCTCGTGCTGGACCAGCTGCGCTGC
-T-T
G-T-A-AT-T-A-C-A-C-A-T
EU 2032 -T
AspLuAupHutPiheGloGlThrHuetGiuAlaHetArgliiHietGlyloPruGIuGlu 334
ECU 1012 -ATTC-TCAA-TAAA-AG-A-TTCA-G-A-T-T-A-T-TTCT- l1LeuSerHlet - Lys - Val - Sir - - - Phi - - - Ser - 357
-
-
GliThrTyrLiLuuG1isLysSirArgAil IiArgGIiAiiLyuGiuGliAr,ThrPha 274
943 GACAAGGACATGTTCCAGGAGACCATGGAGGCCATGAGGATTATGGGCATCCCAGAAGAG
A-T-AT
A-A-CAC-A
52
MICA 93
T-T-CC-T-A
- -- - - PhuSerHis - 337
At.Ies
MICA
-
Anal IAI&TbhrLiuLnNHis6nB SrSirAspLysPhiVnlSmrGliLiuTrpLyoAip
AACATCGCCACACTGCTCCACCAGTCCTCTGACAAGTTTGTCTCGGAGCTGTGGAAGGAT
GB-A -T -AC-TT-G
A-A-GA
-G-B
- VaI - - - - - - - - - Arg - - Ala - - - - VnlAnpArglili GlyLneuAspGiiVniAinGiyHMitSerGiThrAlnLeuProGly
GTGGACCGCATCATCGGCCTGGACCAGGTGGCCGGCATGTCGGAGACCGCACTGCCCOGG
T-G-G-T
T-A-CA-T-T A-T-A-T T-TGG-TCC
- - - - VaI - - Tr -Thr - - - PbhiGlSir
- -
MICA
-
1723 GGCAASGTGGATTACAAAGCTGACGAGTGGCTGATGAAGAACATGGATCCCCTGAATGAC
1732 -G
C-T-G-A-T
T
C
1783
237
PhiGlyLyVPhelliArglIeAuiPheAspValAsoGlyTyrl Va1GlVAIuAsulie 254
MIA
-
IAipTyrLyuAiaAup6i lTrpLimlMhlLyuAuiMitAspProLeuAsnAsp 594
61l1LGVy
luLuiGi1AiuPheGlVAuiAloLysThrViaLyuAunAspAsnSerSurArg 234
643 AACCCCATCCTGGAGGCCTTCGGCAACGCCAAGACCGTGAAGAATGACAACTCCTCCCGC
ECU 652 -T-A-T-C-AT-A-T-A-T-G-T
A-T
A-T-T
-
-
-
ElGB 1972 -
-
H isLysSirLyiLyiAp
AsiPrl
-
6111
GIGlylGiGTrpAuiPbelleAupPhiGlyLeumApLiiGliPriCyullAspLea 514
-
MA
477
LysPbuhiGi1LysPriLys61iLiuLyuAspLysAlaAupPhiCyul lIeI I sTyrAla 574
1993 AAGTTCCAGAABCCCAAGCAGCTGAAGGACAAAGCTGATTTCTGCATTATCCACTATGCC
T
T
A-T-A
A-TCGA-AT-A-A
GCTGGCAAGACGGAGAACACCAAGAAGGTCATCCAGTATCTGGCGTACGTGGCGTCCTCG
A-T-TC-T-C C-T-T-T-T-A
G- A-A-T-A
-H
-
PruLVsAIiTbrAspLysSerPhiValGluLyuVailetGIiGimGnGl yTbrHiiPri 554
1903 CCCAAAGCCACCGACAA6AGCTTCGTGGAGAAGGTGATGCAGGAGCAGGGCACCCACCCC
TA-T-A-C-T-T-A-AC-B-T-A--- A-TT
UC4 1612 -T
- - - LuiVil - - - - Ser - Ser 557
- Thr -
TACAGGAGTATGATGCAAGACCGAGAAGATCAATCCATCTTGTGCACTGGTGAATCTGGA
AT-C-C-T
G6
G-A-T
-T-T-G-C-G-A-TC-T
- Cys LIe.-.------
.
-
MIC 1GiLysPriAi*G6yProProilyliiLuAlualeulemAipG1uGliCysTrpPhi 534
1543 ATTGAGAAGCCAGCAGGCCCCCCGGGCATTCTGGCCCTGCTGGACGAGGAGTGCTGGTTC
1552 -A- GA-T-GAA-T-T-TG-A
TT-T-A-A
- - Ar, - - Asm- - - Val - - - - - - - - - - 537
-
IMIA
-
-
1483 GAGGGCATCGAGTGGAACTTCATCGACTTTGGCCTCGACCTGCAGCCCTGCATCGACCTC
A
A
T-C-G-G-TEUC 1492 -A
-
TyrLynGlyLysLyiArr*6isGuMitPriPriHil hIITyrAliaiIThrAspThrAii 157
TACAAGGGCAABAABAGGCACGAGATBCCCCCTCACATCTATGCCATCACAGACACCGCC
-GA-6
- Arp -
ECU
-
MIA"
LysLouGIuGiuL6ouPhmAuHi sThrMtPbe I LeoGIGu6InGuGiuTyrGInArg 494
1423 AAGCTGCAGCAGCTCTTCAACCACACCATGTTCATCCTGGAGCAGGAGGAGTACCAGCGC
6
T
A
MI4 1432
A-A-A-
-
A
T
tGlyPhBG6 l iPbuAspLuuAuuSurPhul.GhGiLuCyul IuAomTyrThrAumGlu 474
1383 GGCTTCGAGATCTTTGATCTGAACTCGTTTGAGCAGCTGTGCATCAATTACACCAATGAG
A-A-T
C
C
MUC 1372 -A-T-A-T-G
-
ElLA
T
- Ar.
- - - -
ICGA
-
LouHiiAuuLeuLyiGsiAr TyrTyrSerGlyL6el i1TyrThrTyrSurGluLniPhi 117
ElA
-
1303 AAGGCTCTGGACAAGACCAAGAGGCAGGGCGCCTCCTTCATCGGGATCCTGGACATTGCC
A
T-T
-A
-
-
LyuAlhuLuAupLyuThrLyuArgG6luGyAlSurPhmullGlylhuLuuAspluAlo 454
CTGGTGGAGAATGGGAAGAAGGTGAAGGTGAACAAGGATGACATCCAGAAGATGAACCCG
ECU
L
-
-
Arg
-
-
-
.97
AsiGlyViLiiGuGGlyl leArg II CyiArgGliGiyPhuPreAiiArgVnIVolPhe 714
2983 AACGGTGTTCTCGAGGGCATCCGTATCTGCCGCCAGGGCTTCCCCAACAGGGTGGTCTTC
EU 2092 -T--C-G-A-G
A
T
T-C-AA-A-T.__
717
__-IIie
E1LA
Gin
2143 CAB
MUC 2152 -
FIGURE 2. Nucleotide and amino acid sequence comparison between two human nonmuscle myosin heavy chain (NMMHC)
isoforns. Identical amino acids and nucleotides are indicated by dashes. Note that the extra three amino acids in NMMHC-B after
amino acid 203 are responsible for the difference of three in the numbering of subsequent amino acids. The last amino acid for
NMMHC-A (glutamine 715) is the first amino acid of the sequence reported by Saez et al.17
start codon (ATG) is indicated by a 1. The sequence
of 19 nucleotides 5' to the ATG codon has been
confirmed by sequence analysis of a genomic clone
(Simons, Weir, Adelstein, unpublished data), and the
cDNA sequence is shown in Figure 2. The ATG
codon is preceded by an in-frame stop codon located
six nucleotides upstream. The sequence around the
5' ATG start codon bears close similarity to the
Kozak consensus sequences.42
To complete the nucleotide and amino acid sequence between that portion of the human NMMHC
encoded by 4302 and the portion encoded by the
cDNA clones that have been reported previously for
a human NMMHC'7 (see Figure 1), we screened a
Stratagene lambda ZAP T lymphocyte library with a
cDNA probe from the 3' end of 4302 as well as a
cDNA probe from the 5' end of clone HL-1. HL-1 is
a 1.9 kb cDNA clone isolated from the same library
534
Circulation Research Vol 69, No 2 August 1991
4-
(n
m m
W) tt
cK
I
kb
9.5_
Human 7.5302
4.4-
W
V
9.5Human
707
7.5 -*
4.4-
v-
I
'i
m
-~~~~~~~~~~~~~~
,,
-;-
-
m
2~ ~
-8
kb
9.5Human 7.5302
4.4-
S
0
0
E
M
c
cn
I
m
c
e >
-
t~
w
a,
0
,
<-28S
9.57.5-
-.0
Human
707 4.4-
-28S
'9
9~~~~~~~~~~~
-28S
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Eth. Br.
Eth. EBr.
-18S
<-18S
as (4302 and encoding the same portion of the
NMMHC as that encoded by the 5' portion of
pNMHCM2.16 Two clones, (Z801 and ¢Z802, were
isolated and partially analyzed to complete the missing sequence (see Figure 2).
In the course of rescreening the human T lymphocyte lambda gtlO library with an 839 bp chicken
intestinal epithelial cell NMMHC probe (encoding
amino acids 310-590, see Reference 16), five cDNA
clones ((707, 756, 758, 759, and 760, Figure 1) were
isolated. Sequence analysis showed that these clones
encoded a NMMHC isoform distinct from that represented by the clone 0302 and suggested that,
similar to the findings of Katsuragawa et a115 for
chicken NMMHCs, there existed at least two
mRNAs encoding two different human NMMHCs.
Figure 2 presents a comparison between the amino acid
and nucleotide sequences of the two isoforms, which
0
* 28S
<-28S
FIGURE 3. RNA blot analysis of human cell lines. RNA blots
were probed with clones 4302 (nonmuscle myosin heavy
chain-A) and 0707 (nonmuscle myosin heavy chain-B).
Forty micrograms of total RNA was electrophoresed, blotted,
and hybridized with the indicated probes. Cell lines are listed
on the top of each lane. Two different identically loaded gels
are shown. The specific activity of the two different probes was
approximately the same. The autoradiogram of the blot probed
with clone 4707 (B probe) was exposed approximately five
times longer than that probed with (0302 (A probe). Bottom
panel shows a gel stained with ethidium bromide (Eth. Br.).
The cell lines used, from left to right, were BUD-8 (fibroblast),
Hs294T (melanoma), SK-MEL-2 (melanoma), SCC-15
(squamous cell carcinoma), A-431 (epidermoid carcinoma),
HT-29 (adenocarcinoma), FHs74Int (intestinal epithelial
cell), Jurkat (T-cell leukemia), HuT78 (T-cell lymphoma),
SK-N-SH (neuroblastoma), U-138MG (glioblastoma), and
Hs-683 (glioma).
;
FIGURE 4. RNA blot analysis of rat tissues using human A
and B probes. Approximately 30 pg total RNA was electrophoresed in each lane. The same blot was analyzed with each
probe, indicated to the left of the blot. Bands seen below that
at 7.5 kb most likely are due to cross-hybridization of the
probes with smooth muscle (Intestine), cardiac (Heart), and
skeletal muscle (Skeletal M.) mRNA encoding the respective
muscle myosin heavy chains. Bottom panel shows the gel
stained with ethidium bromide (Eth. Br.).
have designated NMMHC-A and NMMHC-B. The
identity of the amino acids between the two isoforms is
89% (amino acids 58-718) and 74% at the nucleotide
level. The differences are spaced throughout the length
of the sequence.
Figure 3 is an RNA blot analysis of various human
cell lines, including Jurkat cells, hybridized with a
cDNA probe for NMMHC-A (4302) and NMMHC-B
(0707). The stringency of the final wash (0.1 x SSC,
65°C) makes cross-hybridization negligible. The blot
probed with clone (4707 was exposed approximately
five times longer than the blot probed with clone 4302.
In general, cultured cells contain more mRNA encoding the A isoform than the B isoform, though the
amount of NMMHC mRNA varies from cell line to
cell line. Both messages appear to be approximately
7.5 kb. Of note is the relatively weak signal seen in
lane SK-N-SH (neuroblastoma) for the mRNA encoding NMMHC-A and the relatively weak signal seen in
lane HT-29 (adenocarcinoma) for the mRNA encoding NMMHC-B (see "Discussion").
Figure 4 compares NMMHC mRNA levels detected in a number of nonmuscle as well as muscle
tissues. Because human tissues were not readily available, we used total RNA prepared from rat tissues.
The same blot was hybridized with the cDNA probe
for NMMHC-A (4302) as well as a cDNA probe for
NMMHC-B (4707), and the exposure time with (4707
we
Simons et al Nonmuscle Myosin Heavy Chains
TABLE 1. Segregation of Nonmuscle Myosin Heavy Chain-A Gene
With Human Chromosome 22
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Gene/chromosome
Human
%
chromosome
-1Discordancy
+/+
+/-/+
1
16
11
16
52
28
2
13
14
14
54
29
3
18
9
18
50
28
4
22
5
38
30
45
5
18
9
8
60
18
6
23
4
26
42
32
7
12
15
28
40
45
8
17
10
20
32
48
9
19
8
13
55
22
12
10
8
24
15
60
11
16
11
12
24
56
12
18
9
22
46
33
13
10
17
26
42
45
14
13
14
30
38
46
17
15
10
29
39
41
16
12
15
25
43
42
17
18
9
40
28
52
18
18
9
33
35
44
19
16
11
13
55
25
20
20
7
21
47
29
21
21
6
42
26
51
0*
22
27
0
0
68
X
9
31
18
37
42
The nonmuscle myosin heavy chain-A gene (NMMHC-A) was
detected as a 19 kb band in EcoRI digests of human-rodent
somatic cell hybrid DNAs after Southern hybridization with a 350
bp intronic probe (data not shown). No cross-hybridizing rodent
sequences were found under the hybridization conditions used.
Detection of the human band is correlated with the presence or
absence of each human chromosome in the group of somatic cell
hybrids. Discordancy represents presence of the gene in the
absence of the chromosome (+t-) or absence of the gene despite
the presence of the chromosome (-/+); the sum of these numbers
divided by total hybrids examined (x 100) represents percent
discordancy. The human-hamster hybrids contained 28 primary
clones and 14 subclones (19 positive of 42 total); the human-mouse
hybrids represented 13 primary clones and 40 subclones (eight
positive of 53 total).
*Two independent human-hamster hybrids contained spontaneous breaks of chromosome 22 between IGLC (22q1l.1-q11.2) and
PDGFB (22q12.3-q13.1). In one hybrid, the proximal long arm and
IGLC were retained, but the myosin gene was lost, whereas the
distal long arm was retained with NMMHC-A in the other hybrid
that had lost IGLC. These results permit regional localization of
the myosin gene to chromosome 22q11.1-qter.
was only approximately 1.5 times longer than that for
4302. The results show that each probe hybridizes
differently. Despite the fact that some cross-hybridization between isoforms A and B might be expected
under these conditions of stringency (0.5 x SSC,
60°C), it still is apparent that the two mRNAs are
differentially expressed in a tissue-dependent manner.
Moreover, similar results to those shown in Figure 4
also were obtained at a stringency of 0.2x SSC, 600C.
This is similar to the findings using avian tissues.15,43
Specifically, rat intestine and thymus appear to con-
535
tain more mRNA encoding NMMHC-A than mRNA
encoding NMMHC-B, whereas rat brain and testis
appear to be relatively enriched for the mRNA encoding NMMHC-B compared with NMMHC-A. Lung
and kidney appear to contain relatively large amounts
of both mRNAs. Of note are the mRNAs detected at
6.5-6.9 kb in intestine (smooth muscle), heart, and
skeletal muscle cells; these bands presumably are
due to cross-hybridization of the probes with the
differentiated forms of the respective mRNAs for
muscle myosins, which are more divergent from both
NMMHC-A and NMMHC-B than are the A and B
isoforms from each other. The detection of the muscle-specific mRNAs is dependent on both the reduced
stringency of hybridization and the presence of these
mRNAs in levels several orders of magnitude greater
than the concentrations of nonmuscle myosin mRNAs
in these tissues. It also should be noted that tissuespecific expression of NMMHC-A and NMMHC-B
cannot be explained by cross-hybridization between
these two probes but that the extent of tissue-specificity almost certainly is underestimated because of
cross-hybridization.
Mapping Nonmuscle Myosin Heavy Chain Genes
Using Human-Rodent Somatic Cell Hybrids
Both NMMHC genes were chromosomally mapped
by Southern analysis of DNAs isolated from a panel of
human-rodent somatic cell hybrids using 32P-labeled
cDNA and genomic fragments as probes. A 350 bp
genomic intronic probe of NMMHC-A identified a 19
kb band in EcoRI digests of human DNA. No crosshybridization with rodent sequences was found. Analysis of an entire series of human-rodent hybrid cell
DNAs with the 350 bp genomic probe as well as a
second genomic probe (data not shown) permitted
unambiguous assignment of the NMMHC-A gene to
human chromosome 22 (Table 1). The gene segregated discordantly (.18%) with all other human
chromosomes. Examination of two hybrids containing
spontaneous breaks involving chromosome 22 permitted regional localization of NMMHC-A to the region
22q11.1-qter.
A 1.59 kb NMMHC-B cDNA probe (4707) detected six (2.9, 5.7, 8, 10, 16, and 22 kb) bands in
EcoRI-digested human DNA as well as several crosshybridizing sequences in rodent DNAs (data not
shown). In contrast to the NMMHC-A probe, the
NMMHC-B cDNA probe identified hybridizing
bands in an entirely different series of hybrid cell
lines in the same panel, indicating localization of the
gene on a different chromosome. The 2.9, 5.7, and 22
kb bands segregated concordantly (i.e., all present or
all absent) in the hybrids, and they were localized to
human chromosome 17 (Table 2), and there was
greater than or equal to 21% discordancy with all
other chromosomes. Moreover, the same panel of
hybrids was analyzed with a p53 tumor antigen
(TP53) probe, and there was precise correlation
between hybrids retaining the TP53 gene and the
NMMHC-B gene (data not shown). It was possible to
536
Circulation Research Vol 69, No 2 August 1991
TABLE 2. Segregation of Nonmuscle Myosin Heavy Chain-B Gene
With Human Chromosome 17
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Gene/chromosome
Human
/
chromosome
Discordancy
+1
-1+
+1+
I
39
7
17
27
27
43
3
2
23
21
27
33
13
3
22
22
39
20
26
4
32
12
42
5
42
21
4
23
30
6
29
17
30
14
34
7
27
17
8
28
38
33
13
8
23
21
38
39
9
25
19
7
29
41
10
14
30
5
39
11
37
18
26
9
39
12
38
28
16
8
27
43
13
3
16
28
21
14
14
37
30
9
26
15
28
16
15
31
34
32
16
20
24
14
42
1
17
43
1
45
2*
30
18
30
14
16
33
19
22
22
6
40
31
20
25
19
15
31
38
21
35
9
23
23
36
22
17
27
9
37
40
26
18
19
27
41
X
The nonmuscle myosin heavy chain-B gene (NMMHC-B) was
detected as 2.9, 5.7, 8, 10, 16, and 22 kb bands in EcoRI digests of
human-rodent somatic cell hybrid DNAs after hybridization with
the 1.59 kb NMMHC-B cDNA clone 4707. The 2.9, 5.7, and 22 kb
bands cosegregated in all hybrids, and these bands could be
resolved from cross-hybridizing rodent sequences (data not
shown). The human-hamster hybrids consisted of 28 primary
clones and 13 subclones (16 positive of 41 total); the human-mouse
hybrids contained 19 primary clones and 30 subclones (28 positive
of 49 total).
*The two discordancies represent failure of both TP53 and
NMMHC-B to segregate with other chromosome 17 markers in
two independent human-hamster hybrids. In addition, TP53 and
NMMHC-B segregated discordantly with all other chromosome 17
markers in eight independent hybrid lines all isolated from one
series of human-mouse hybrids involving a single human parental
(VA2) cell line (data not shown). These results strongly suggest
that the human parental HPRT- SV40 transformed WI 18 line
VA244 used in preparing this single group of somatic cell hybrids35
contains one copy of chromosome 17 with a break involving 17pl13,
because eight independent hybrids from this series retained chromosome 17 in the absence of both TP53 and NMMHC-B, whereas
four other hybrids retained all chromosome 17 markers including
TP53 and NMMHC-B.
localize the gene regionally by examining another
series of hybrids isolated after fusing human parental
cells containing a reciprocal chromosome 17;22
(p13;qll) translocation with mouse fibroblasts.36
Four independent hybrids retained the 17p13-qter
translocation chromosome, and human TP53 and
NMMHC-B were absent from all four hybrids. Thus,
NMMHC-B can be assigned to band 17pl3, which is
also the locus for TP53.36
t
I
_w
W
j.
4~
_44A
i~~~~
.e
p *.
-
*
#3**
.
4
.A
*
§
*s s
~~*
' f
Ar
21
4}
:9%
*
*
22
t
v
FIGURE 5. In situ hybridization with nonmuscle myosin
heavy chain A cDNA probe. Top panel: Representative
metaphase spread containing a grain on chromosome 22
(lower arrow) and one on another chromosome to indicate
background (see bottom panel). Middle panel: Same metaphase spread after Giemsa replication banding. Bottom
panel: Distribution of grains on chromosome 22 and background grains on chromosomes 21 and Y in 61 metaphases.
Regional Localization by In Situ Hybridization
The presence of NMMHC-A on chromosome 22
was confirmed and the gene was regionally localized
to 22q11.2 by in situ hybridization of metaphase
Simons et al Nonmuscle Myosin Heavy Chains
TABLE 3. Comparison of Chicken and Human Myosin Heavy
Chain Amino Acids (% Identity)
HNMB
CNMB
HNMA
88
CNMA
88
92
CNMB
...
98
79
HNMA
89
...
...
CNMA, chicken nonmuscle myosin heavy chain (MHC)-A.16
Entire amino acid sequence used; CNMB, chicken nonmuscle
MHCGB15; HNMA, human nonmuscle MHC-A (see Figure 2);
HNMB, human nonmuscle MHC-B (see Figure 2).
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
spreads with a 3H-labeled NMMHC-A cDNA probe
(4302, Figure 5). Metaphase spreads containing a
grain on a small (G group) chromosome were chosen
for analysis. Thirty-eight grains (26% of total grains)
of the total 146 grains were found on chromosome 22,
and 12 of the grains (32%) were specifically localized
to band qll.2. The other grains were randomly
distributed on chromosomes 21,Y and other chromosomes (Figure 5C). No clustering of grains was
detected on chromosome 17.
Discussion
The results presented here show that two distinct
200 kDa MHC isoforms exist in human cells and that
these isoforms are encoded by genes located on
different chromosomes. The two isoforms show considerable sequence similarity. Sequence comparison
of human and chicken NMMHC isoforms leads us to
postulate that human NMMHC-A is a homologue of
chicken FMHA, and human NMMHC-B is a homologue of chicken FMHC (Table 3, Reference 15).
Note that both NMMHC isoforms are highly conserved between the species. Indeed, the sequence
identity between the human and chicken NMMHC-A
(92%) and human and chicken NMMHC-B (98%) is
greater than the identity between either the human
or chicken NMMHC-A and NMMHC-B (89% or
88%, see Table 3). Thus, the conservation of sequence between the species is greater than conservation of sequence between the isoforms, which
suggests a distinct function for each isoform. These
distinct functions might be expected to be reflected
by the differential expression of each isoform in
different tissues or cell types. To investigate this, we
performed the series of Northern blot analyses
shown in Figures 3 and 4. In general, the mRNA
encoding NMMHC-A is more abundant than that
encoding NMMHC-B in human cell lines. However,
in agreement with recent studies using avian tissues,
which reported an increase in the mRNA encoding
NMMHC-B in brain,'1543 we show that the mRNA for
B is more abundant than that for A in human
neuroblastoma cells (SK-N-SH). However, the finding that glioma (Hs-683) and glioblastoma (U138MG) cells contain relatively more mRNA encoding NMMHC-A rather than -B suggests significant
differences in the distribution of these isoforms
within brain tissues.
537
Our results with rat tissues, though not quantitative,
support the findings from avian tissues which were
that the mRNA encoding NMMHC-A was relatively
more abundant than the mRNA for NMMHC-B in
spleen and intestinal epithelial cells, whereas the
mRNA encoding NMMHC-B was relatively more
abundant in brain and testis. Kidney contains approximately equal amounts of both mRNAs.
Combining the sequence presented here with that of
Saez et al17 gives a protein sequence for NMMHC-A of
1,961 amino acids, which is two amino acids more than
the 1,959 amino acids reported by Shohet et al16 for the
chicken NMMHC-A. This discrepancy results from
an insertion of an extra glutamine residue within the
run of five glutamine residues at amino acids 1,3461,35016 and an insertion of alanine and serine for a
single valine residue at amino acid 1,390.16 Also, all
NMMHCs sequenced to date have a deletion in the
area of amino acid 202 compared with the smooth
muscle MHC,45 the significance of which is presently
unknown. NMMHC-A has a deletion of 10 amino
acids, and NMMHC-B has seven amino acids deleted.
Cloned cDNA and genomic probes of NMMHC-A
and NMMHC-B have been used to localize these
genes to human chromosomes 22 and 17, respectively, by Southern analysis of DNAs from a panel of
human-rodent somatic cell hybrids. Furthermore, the
gene for NMMHC-A has been localized to a region
22q11.2 by in situ hybridization of metaphase chromosome spreads. The NMMHC-B gene was localized
further to a region 17p13 by analysis of hybrids
constructed from parental cell lines containing a
translocation involving this telomeric band. The extent of cross-hybridization between NMMHC-A and
NMMHC-B, under conditions of high stringency, was
negligible and therefore allowed localization of the
NMMHC-B gene using the corresponding cDNA
probe. The localization of the NMMHC-A gene to
chromosome 22 is in agreement with the findings of
Saez et al.17 No subchromosomal localization was
reported in that study.
The gene for NMMHC-B was localized to a region
of chromosome 17 known to carry three sarcomeric
(skeletal) MHC genes (MYH 1, 2, 4)26-30 and a fetal
skeletal MHC gene (MYH 3).27 A gene for the atrial
(cardiac) myosin light chain (MYL 4)46 also is present
on that chromosome. A fifth skeletal MHC gene
(MYH 5) has been localized provisionally on chromosome 7 by in situ hybridization with a mouse conserved
3' cDNA coding sequence.47 Two cardiac MHC genes
are located on chromosome 14.48 49The significance of
the localization of one of the NMMHC genes to the
same area of chromosome 17 that contains skeletal
MHC genes is unclear, because these represent members of two multigene families that diverged before
speciation, and more closely related MHC genes of
each type also are found on entirely different chromosomes. Nevertheless, the probability of random association of two genes in a region representing no more
than 0.5% of the human genome is not high.
538
Circulation Research Vol 69, No 2 August 1991
It is safe to assume that the nonmuscle isoforms
are the oldest myosins, whereas more specialized
proteins such as skeletal and cardiac myosins developed later in the course of evolution. Furthermore, a
very significant sequence conservation between cor-
responding chicken and human NMMHCs implies
that two nonmuscle myosins arose relatively early.
The localization of the NMMHC-B gene to the same
area of chromosome 17 as skeletal MHC genes
suggests that this gene may have been the ancestor of
skeletal muscle myosin genes. The availability of
cDNA clones encoding human NMMHC isoforms
will allow detailed study of their function and regulation and should help shed light on the role played
by nonmuscle myosins in various biological processes,
such as smooth muscle proliferation.
Acknowledgments
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
The authors gratefully acknowledge the expert
technical assistance of Yvette A. Preston and Judy
Yu and the expert manuscript preparation of
Catherine Magruder.
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18. Ross R: The pathogenesis of atherosclerosis: An update. N
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KEY WORDS * gene localization * cDNA cloning *
expression
gene
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Human nonmuscle myosin heavy chains are encoded by two genes located on different
chromosomes.
M Simons, M Wang, O W McBride, S Kawamoto, K Yamakawa, D Gdula, R S Adelstein and L
Weir
Circ Res. 1991;69:530-539
doi: 10.1161/01.RES.69.2.530
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